Volume 2: Science
5. Diagnosis and therapy
Diagnosis
Developments in post-mortem diagnosis of BSE
Developments in ante-mortem diagnosis of BSE
Diagnosis of vCJD

5.18 Since its emergence, BSE has been diagnosed from clinical observations and confirmed by detailed histopathological examination of the brain following slaughter of the animal. The histopathological examination is based on the detection of characteristic vacuolation (sponginess) affecting the grey matter of the brain. This vacuolation in BSE is orderly, and usually distributed in a symmetrical bilateral fashion at the obex (see the full colour illustrations in vol. 16: Reference Material). The major target sites for examination are in the medulla oblongata of the brain, the lowest part of the brainstem, next to the spinal cord. ¹

5.19 Although significant advances have been made in the development of diagnostic tools since the emergence of BSE, clinical observations and histopathological examination of the brain are still the chosen form of diagnosis. Only recently has an ante-mortem test been developed which shows promise for the early diagnosis of BSE from peripheral blood samples. ² An alternative method is the DELFIA(R) technique, which is a fluoroimmunoassay for the analysis of PrP^c in human blood and its components; PrP^c was found mostly in plasma (68.5 per cent) and platelets (26.5 per cent). ³

Developments in post-mortem diagnosis of BSE

5.20 In 1987 two techniques for diagnosing TSEs had been described and were in use at the Central Veterinary Laboratory (CVL). The first involved histopathological examination of brain tissue as described in paragraphs 3.6-3.10. The second technique was a test for scrapie-associated fibrils (SAFs) which detected the presence of SAFs in processed homogenates of brain tissue by electron microscopy.

5.21 Earlier, during 1985 and 1986, Dr Harash Narang and co-workers had developed a third diagnostic test for spongiform encephalopathies, in particular scrapie. The technique involved taking brain slices and placing carbon-coated grids onto the cut surface of the section. ⁴ The grids were then soaked in a protein denaturing solution, fixed, stained and observed under an electron microscope. Positive diagnosis was made on detection of the presence of tubulofilaments, structures similar to but discernible from normal cell tubules. Dr Narang's technique was likened to the 'touch' or 'impression' techniques used in haematological and other diagnostic studies. It was more rapid than the CVL's SAF test, though required material taken straight from the brain. In contrast, the CVL's SAF test used homogenised brain and was thus more suitable for macerated brains, which were unsuitable for histopathology. Dr Narang's test, however, was not considered to have sufficient sensitivity and specificity to replace other methods. The work of Dr Narang and the appraisal of his touch test is considered in vol. 11: Scientists after Southwood.

5.22 As discussed previously (see paragraphs 2.68-2.69), SAFs were found to be composed of the protease-resistant prion protein, PrP^Sc. ⁵ PrP^Sc became a marker for TSEs in general, and BSE in particular, and opened up a new avenue for research into a diagnostic test. Again it is helpful to note events after 1996. Several tests have been developed based on this marker by many groups, including the Neuropathogenesis Unit (NPU) and CVL, but progress has been slow. In July 1999, four tests were selected and evaluated by the European Commission. ⁶ The companies involved were:

1. E.G. & G. Wallace, UK;
2. Prionics A.G., Switzerland;
3. Enfer Technology Ltd, Ireland (licensee of Proteus International plc); and
4. Commissariat à l'Energie Atomique (CEA), France.

5.23 All four tests involved the detection of protease-resistant PrP^Sc in brain and spinal cord homogenates from confirmed BSE cases using material from healthy New Zealand cows as controls. The results indicated that post-mortem tests ii., iii. and iv. have 'excellent potential' for confirming clinical BSE or for rapidly screening dead or slaughtered animals. ⁷ Indeed, the Prionics test is currently being used routinely in Switzerland to detect BSE-infected animals at the abattoir to ensure that meat reaching the market is BSE-free. Validation of the Prionics test has recently been carried out using BSE cattle and controls. The authors observed that the test was highly sensitive, specific and reliable, and that sampling the correct area of the brain and the method of protein extraction are important for accurate diagnosis. Subclinical cases were also identified using this test. The authors concluded that it was a useful procedure for surveillance of cattle at the slaughterhouse. ⁸

Developments in ante-mortem diagnosis of BSE

5.24 Work on ante-mortem tests began in the early 1990s. In 1991, Mr Roy Jackman of the CVL began development of the Electrochemical Urine Test for identification of BSE in cattle. This had initially been used to test scrapie-infected sheep. The method detected three urine metabolites, uric acid, catechol sulphate and an unknown, whose concentrations could be used diagnostically for BSE. However, false positive results were obtained in the initial trials. ⁹

5.25 Further work, in an attempt to improve this test, took place in collaboration with Professor Barritault's group in Paris, starting in August 1996. By 1998, the metabolite Marker T (thought to be the unknown marker previously identified at the CVL) had been isolated and identified in the urine of BSE-infected cattle. Plans have been made to characterise this metabolite and identify others, and to attempt to detect a marker in blood. ¹⁰

5.26 The four tests evaluated by the European Commission in 1999 are able to detect low levels of PrP^Sc. This suggested that these tests could be used to detect infection in tissues or fluids before the development of clinical signs. However, there is little information available on the relationship between infectivity titres and PrP^Sc concentrations in different tissues and fluids throughout the incubation period. Furthermore, for a useful ante-mortem test, detectable levels of infectivity must be obtained in easily accessible fluids or tissues.

5.27 Both before and since March 1996, several protein markers for BSE other than PrP^Sc, such as 14-3-3 protein and S100 protein, have been detected in the cerebrospinal fluid (CSF) of BSE-affected cattle. ¹¹ These proteins are thought to originate from neuronal tissue and appear in the CSF as a result of the disease. However, none of the tests for these markers is sufficiently reliable for the diagnosis of TSEs.

5.28 In 1999 a promising test using capillary immunoelectrophoresis (CIE) was introduced for ante-mortem detection. It is claimed that the test can identify TSE agent in the blood of hamsters four weeks after parenteral inoculation. Also, white blood cell preparations (buffy coats) from lambs born of scrapie-affected ewes have been found to contain abnormal PrP one month after birth using the CIE test. The test depends on the complete removal of PrP^C by enzyme digestion; incomplete removal can lead to false positive results. The CIE test is currently undergoing further evaluation. Another recently developed ante-mortem test for PrP^Sc is based on a paraffin-embedded tissue blot, which has been shown to detect disease in infected mice 30 days after inoculation and 145 days before the onset of clinical signs. ¹²

Diagnosis of vCJD

5.29 Diagnosis of vCJD is initially based on clinical information (see vol. 8: Variant CJD). The clinical presentation of the disease is with behavioural and psychiatric disturbances. Patients sometimes complain of feeling extremely cold and of experiencing attacks of pain in the lower limbs. After several weeks or months, motor problems develop, and muscle coordination and balance maintenance are affected. This is followed by dementia, muscle paralysis and mutism. Other important information for diagnosis is the young age at onset and the prolonged clinical course. The median age in sporadic CJD at death is 66 years, compared with 29 years for vCJD. ¹³ The median illness duration from the first symptom to death is 4 months in sporadic CJD but 14 months for vCJD.

5.30 The diagnosis is then confirmed by ante-mortem histopathological examination of a brain biopsy or by post-mortem histopathological brain examination. These examinations aim to detect the characteristics of vCJD: 'florid plaques' in the cerebrum and cerebellum, abundant PrP deposition, spongiform change (especially in the basal ganglia) and severe glial cell ¹⁴ proliferation in the thalamus. ¹⁵

5.31 However, the ante-mortem examinations of brain biopsies are not thought to be reliable. In oral evidence to the Inquiry, Dr Robert (now Professor) Will of the CJD Surveillance Unit (CJDSU) explained that the problem with biopsies is that only a very small piece of brain tissue is removed which may be 'from an area of the brain that is not affected fully by the pathological process, and indeed you may sometimes sample an area that is normal, which may subsequently become abnormal. So a negative test does not exclude a diagnosis.' ¹⁶ There are also inherent risks in the brain biopsy procedure, which means that it is only performed in the very late stages of disease. Therefore, the development of a reliable ante-mortem test for early diagnosis of vCJD has been considered to be very important.

5.32 Many of the tests already described for ante-mortem BSE diagnosis have been examined for their use in CJD diagnosis. Dr Narang reported detection of SAFs in the urine of patients with CJD but was unable to distinguish between sporadic and variant CJD. ¹⁷ In 1997 the MRC funded an 18-month long evaluation at the CJDSU of Dr Narang's urine test, which involved concentrating urine several thousand times and detecting 'nemavirus' particles (see paragraphs 2.59-2.61) using electron microscopy. ¹⁸ Many problems were associated with the study and at the end of the 18-month period, no conclusions on the test's reliability had been reached (see vol. 11: Scientists after Southwood). ¹⁹

5.33 It was shown in work published in September 1996 that sporadic CJD could be diagnosed by the detection of 14-3-3 protein in CSF. ²⁰ However, it has not proved to be so useful in the diagnosis of the slowly progressing vCJD. More recently, the German National CJD Surveillance Unit suggested the measurement of S100 protein in serum as an early ante-mortem test for vCJD. ²¹ However, it was not found to be sufficiently specific for CJD. ²² Other protein markers studied by the German group were tau protein and neuron specific enolase (NSE). ²³

5.34 Neuro-imaging techniques, such as single photon emission computed tomography (SPECT) analysis and magnetic resonance imaging (MRI) have also been recently proposed. These techniques are used when patients present with clinical symptoms suggestive of a neurological disorder. A reduction in the blood flow to the brain was detected in patients with vCJD by SPECT analysis. ²⁴ Although this finding was again not specific to CJD, it may help to raise initial suspicion of the possibility of this disease. Recent MRI studies in cases with suspected vCJD have revealed specific changes in the posterior thalamus (pulvinar) during the course of the clinical disease in 78 per cent of 36 confirmed cases and in none of 57 controls. The test promises to be of considerable value in the differential diagnosis of suspected vCJD. ²⁵

5.35 Studies carried out by Professor John Collinge's group in 1999 indicate that detection of protease-resistant PrP^Sc in tonsil or appendix biopsy samples may provide a diagnostic tool for early ante-mortem identification of CJD. ²⁶ This process utilises the finding in animal studies that infectivity replicates in lymphoreticular tissue, including the tonsils, before neuroinvasion. ²⁷ Variant CJD can be distinguished from sporadic CJD by examining the glycoform patterns of PrP^Sc detected in the tissue sample (see paragraphs 4.8-4.9). These techniques are now being employed in the anonymous testing of tonsil and appendix tissue in three centres in the UK, in an attempt to determine the prevalence of vCJD (see paragraphs 4.22-4.23). ²⁸

5.36 With the increasing confirmation that the lymphoreticular system (LRS) is involved in agent replication comes the concern that infectivity could be present in the human bloodstream, suggesting that blood transfusions could therefore be a route of transmission. Although blood from guinea pigs and mice infected intracerebrally with CJD has been shown to be infective (paragraph 2.134), there is to date no evidence of disease transmission in humans from blood transfusions or blood products. ²⁹ However, BSE has been transmitted recently to a sheep via transfusion of 400 ml of blood. It is important to note that the blood came from an apparently healthy sheep, to which an oral dose of BSE had been administered. The donor sheep was in fact half way through its incubation at the time the blood was taken. ³⁰ Like patients with vCJD, infectivity in BSE-infected sheep is present in the LRS and so may provide a suitable model for assessing the risks of infection from human blood transfusion and also for evaluating preventive measures such as leuco-depletion. This report gives support to suggestions that there may be risks from blood transfusions and emphasises the importance of developing a diagnostic blood test for vCJD. The Department of Health (DH) has initiated research in this area, under the leadership of Dr Eglin (Public Health and Laboratory Service, Leeds). ³¹ Leucodepletion (removal of white cells) is currently being used to reduce the risk of transmission by blood transfusion.

5.37 Early detection of TSEs is important both for preventing disease transmission and for potential treatment. So far, the tests described above have only assayed a small number of samples, but with further study a specific and sensitive test may be obtained.